Dnase

Tumors isolated from mice during above-described biodistribution experiments were then used for flow cytometry studies of polyplex uptake. Tumors were cut into small pieces, washed with HBSS containing Ca²⁺ and Mg²⁺, and then processed using an enzyme mix containing collagenase (0.5 mg/mL, Roche Life Sciences, Indianapolis, IN, USA) and DNase (0.19 mg/mL, BioRAD, Hercules, CA, USA) in DMEM. After 1 h incubation in the enzyme mix, the tumors were centrifuged and resuspended in HBSS without Ca²⁺ and Mg²⁺ and then incubated with 5 mM EDTA for 20 min. Tumors were then centrifuged, and the pellets were resuspended in HBSS with Ca²⁺ and Mg²⁺ and filtered using a 70 μm Nylon cell strainer. Filtrate was then washed once more with HBSS containing Ca²⁺ and Mg²⁺ and then incubated in ACK lysis buffer (Thermo Fisher Scientific, USA) for 2 min before being diluted in 20 mL of PBS−/−. Cells were then pelleted and resuspended in 1–2 mL PBS−/− prior to running on a flow cytometer (BD LSRii, BD Biosciences, San Jose, CA, USA). Uptake analysis was performed in FlowJo. Cell populations were isolated using forward and side scatter, then GFP positive tumor cells were gated, and Cy5 fluorescence intensity was measured for the GFP positive tumor cell population.

RNA was extracted using Tri-Reagent (Sigma Aldrich) according to the manufacturer’s instruction and residual genomic DNA was removed by DNase (Bio-Rad, Hercules, CA USA) treatment. RNA was quantified by determining 260/280 using a spectrophotometer (Molecular devices, Ramsey MN USA). Microsome and cytosol fractions were prepared by homogenization with a dounce homogenizer followed by differential centrifugation as previously described [42 (link)]. Protein concentrations were determined with Bradford reagent (Bio-Rad).

Intracellular delivery of fluorescent si-NPs was evaluated by flow cytometry. Tumors were with collagenase (0.5 mg/mL, Roche Life Sciences) and DNAse (0.19 mg/mL, BioRAD) for 1 h, incubated with 5 mM EDTA for 20 min, re-suspended in HBSS and filtered through a 70 μm nylon cell strainer. Erythrocytes were removed with ACK lysis buffer (Thermo Fisher Scientific) for 2 min, and washed cells were assessed by flow cytometry (BD LSRii, BD Biosciences, San Jose, CA, USA). Uptake analysis was performed in FlowJo. Cell populations were isolated using forward and side scatter, then GFP positive tumor cells were selected, and Cy5 fluorescence intensity was measured.